Sustained exposure and adherence to antiretroviral therapy (ART) are the main predictors of efficacy in HIV treatment. Although suboptimal adherence (e.g., less than daily dosing) can achieve viral suppression by conventional assays, it is unclear whether variable ART adherence has any impact beyond HIV suppression. In this proposal we aim to determine the impact of ART adherence on biological outcomes such as the HIV reservoir, immune activation and chronic inflammation in suppressed, HIV-infected individuals. Based on its uniquely long half-life (~17 days) in dried blood spots (DBS), we will utilize tenofovir-diphosphate (TFV-DP) as a novel biomarker to objectively quantify cumulative drug adherence and to correlate it with the size and dynamics of the viral reservoir and the levels of immune activation and chronic inflammation. Herein, we hypothesize that, in virologically-suppressed, HIV-infected individuals on chronic ART, suboptimal adherence and exposure (measured using TFV-DP in DBS) will be associated with higher levels of residual viremia and a larger HIV reservoir (measured by levels of cell-associated RNA [CA-RNA] and CA- DNA) in CD4+ T cells. In addition, we further hypothesize that suboptimal ART adherence will also be associated with increased immune activation and chronic inflammation. To test our hypotheses we propose the following aims. Aim 1: Initially, in this aim we will quantify the levels of CA-RNA, CA-DNA, and plasma residual viremia in suppressed, HIV-infected individuals and correlate them with levels of TFV-DP in DBS as a measure of cumulate drug exposure (adherence). Subsequently, we will evaluate the changes in the HIV reservoir in relation to changes in ART adherence over time. Aim 2: This aim will investigate if the levels of residual CD8+ and CD4+T cell immune activation, inflammatory cytokines and soluble markers in suppressed, HIV-infected individuals are associated with variations in ART drug adherence (measured by TFV-DP levels in DBS) and with the HIV reservoir measures quantified in Aim 1. In this R21 application, we challenge the current paradigm that suppressive ART adherence is the ultimate clinical goal to prevent HIV disease progression. Instead, we propose that suboptimal adherence (measured with an objective biomarker) has deleterious biological consequences, even if it is sufficient to maintain viral suppression. Understanding the relationship between suboptimal adherence, HIV persistence and chronic inflammation is highly clinically relevant, as it could help optimize ART and maximize the effect of HIV cure and eradication strategies.